Identification of active flavin-containing monooxygenase isoform 2 in human lung and characterization of expressed protein.

Full-length human (hFMO2.1) and monkey (mFMO2) flavin-containing monooxygenase proteins, which share 97% sequence identity, were produced by baculovirus-mediated expression in insect cells and assayed for S-oxygenation under conditions known to affect FMO activity. Both enzymes demonstrated maximal activity at pH 9.5; but hFMO2.1 retained significantly more activity than mFMO2 did at pH 9.0 and higher. hFMO2.1 also retained significantly more activity than mFMO2 did in the presence of magnesium and all detergents tested. Although hFMO2.1 had more residual activity after heating at 45 degrees C than mFMO2, under some conditions, both had less than 10% of control activity, whereas expressed rabbit FMO2 retained over 50% activity. Screening for NADPH-oxygenation by hFMO2.1, indicated that substituted thioureas with a small cross-sectional area (2.4-4.3 A) are good substrates, whereas 1,3-diphenylthiourea (11.2 A) was not oxygenated. We confirmed the presence of hFMO2.1 in lung tissue from a heterozygous individual (hFMO2*1/hFMO2*2A) by Western analysis and confirmed activity by S-oxygenation. These microsomes also demonstrated a heat-associated loss of activity similar to expressed hFMO2.1. The heat sensitivity of hFMO2.1 may partially explain why activity in post mortem human lung samples has previously been unreported. Individuals that have the FMO2*1 allele-encoding full-length hFMO2.1 may exhibit altered drug metabolism in the lung.     

Genetic polymorphisms of flavin-containing monooxygenase (FMO)

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU). The TMAU patient excretes large amounts of TMA in urine and sweat. A more recent ethnically related polymorphism in expression of the major FMO in lung, FMO2, has been described. All Caucasians and Asians genotyped to date are homozygous for a CAG --> TAG amber mutation resulting in a premature stop codon and a nonfunctional protein truncated at AA 472 (wildtype FMO2 is 535 AA). This allele has been designated hFMO2*2A. Twenty-six percent of individuals of African descent and 5% of Hispanics genotyped to date carry at least one allele coding for full-length FMO2 (hFMO2*1 allele). Preliminary evidence indicates that FMO2.1 is very active toward the S-oxygenation of low MW thioureas, including the lung toxicant ethylene thiourea. Polymorphic expression of functional FMO2 in the individuals of African and Hispanic descent may markedly influence drug metabolism and/or xenobiotic toxicity in the lung.     

pH dependence on functional activity of human and mouse flavin-containing monooxygenase 5

Flavin-containing monooxygenase (FMO) 5 belongs to a family of enzymes that catalyze the oxygenation of nucleophilic N- and S-containing compounds. The FMO enzyme family consists of five forms (FMOs1-5) that share about 50-60% sequence identity to each other. A comparison of FMOs showed that the pH-dependence profile for functional activity of FMO5 differed significantly from that of other FMO enzymes. The objective of this study was to examine the pH-dependence of FMO5 to gain insight into the mechanism of action of FMO5. Recombinant mouse and human FMO5 (mFMO5 and hFMO5, respectively) were expressed as maltose-binding fusion proteins from Escherichia coli, purified with affinity chromatography, and examined for their N-oxygenation functional activity at different pH values. hFMO5 showed a broader range and greater functional activity from pH 6 to 11 compared to mFMO5. mFMO5 lost almost all functional activity at pH 6, while hFMO5 maintained almost normal enzyme activity. In order to identify the amino acid residues involved in the effects of pH on hFMO5 and mFMO5 functional enzyme activity, pH-studies in the range of pH 6-9 were done with chimeras of recombinant mouse and human FMO5 and variants of both. Results of these studies and molecular modeling showed that residues responsible for the differences in the pH profile between mFMO5 and hFMO5 were located at positions 227 and 228 of the enzyme. Further variants were made to investigate the role of these amino acids. The results of this study may help to explain the mechanism of FMO function.     

Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes

The stereoselective sulfoxidation of the pharmacologically active metabolite of sulindac, sulindac sulfide, was characterized in human liver, kidney, and cDNA-expressed enzymes. Kinetic parameter estimates (pH = 7.4) for sulindac sulfoxide formation in human liver microsomes (N = 4) for R- and S-sulindac sulfoxide were V(max) = 1.5 +/- 0.50 nmol/min/mg, K(m) = 15 +/- 5.1 microM; and V(max) = 1.1 +/- 0.36 nmol/min/mg, K(m) = 16 +/- 6.1 microM, respectively. Kidney microsomes (N = 3) produced parameter estimates (pH = 7.4) of V(max) = 0.9 +/- 0.29 nmol/min/mg, K(m) = 15 +/- 2.9 microM; V(max) = 0.5 +/- 0.21 nmol/min/mg, K(m) = 22 +/- 1.9 microM for R- and S-sulindac sulfoxide, respectively. In human liver and flavin-containing monooxygenase 3 (FMO3) the V(max) for R-sulindac sulfoxide increased 60-70% at pH = 8.5, but for S-sulindac sulfoxide was unchanged. In fourteen liver microsomal preparations, significant correlations occurred between R-sulindac sulfoxide formation and either immunoquantified FMO or nicotine N-oxidation (r = 0.88 and 0.83; P < 0.01). The R- and S-sulindac sulfoxide formation rate also correlated significantly (r = 0.85 and 0.75; P < 0.01) with immunoquantified FMO in thirteen kidney microsomal samples. Mild heat deactivation of microsomes reduced activity by 30-60%, and a loss in stereoselectivity was observed. Methimazole was a potent and nonstereoselective inhibitor of sulfoxidation in liver and kidney microsomes. n-Octylamine and membrane solubilization with lubrol were potent and selective inhibitors of S-sulindac sulfoxide formation. cDNA-expressed CYPs failed to appreciably sulfoxidate sulindac sulfide, and CYP inhibitors were ineffective in suppressing catalytic activity. Purified mini-pig liver FMO1, rabbit lung FMO2, and human cDNA-expressed FMO3 efficiently oxidized sulindac sulfide with a high degree of stereoselectivity towards the R-isomer, but FMO5 lacked catalytic activity. The biotransformation of the sulfide to the sulfoxide is catalyzed predominately by FMOs and may prove to be useful in characterizing FMO activity.     

Differences in FMO2*1 allelic frequency between Hispanics of Puerto Rican and Mexican descent.

A polymorphism for the phase I drug-metabolizing enzyme, flavin-containing monooxygenase isoform 2 (FMO2), encoding either truncated inactive protein, FMO2X472 (FMO2.2A), or full-length active enzyme, FMO2Q472 (FMO2.1), is known and exhibits significant interethnic differences in allelic frequency. FMO2 is the major or sole FMO isoform expressed in the lung of most mammals, including nonhuman primates. To date, FMO2.1 has been found only in African-American and Hispanic populations, rendering individuals with this allele subject to drug metabolism that is potentially different from that of the general population. Approximately 26% of African-Americans (n = 180) possess the FMO2*1 allele. In preliminary studies, we initially estimated that 5% of Hispanics (n = 40) have the FMO2*1 allele, but access to large cohorts of individuals of defined national origin has allowed us to determine the occurrence among Mexican-American and Puerto Rican-American groups. We used allele-specific genotyping to detect FMO2*1 from 632 Hispanic individuals, including 280 individuals of Mexican origin and 327 individuals of Puerto Rican origin. Statistical analysis indicated that results from Mexican (five sample sources) and Puerto Rican (three sample sources) samples were consistent with the hypothesis of homogeneity within each group from different sources. Data were subsequently pooled across sources to test for evidence of a difference in occurrence of FMO2*1 between ethnic groups. There was strong evidence (p = 0.0066) that FMO2*1 is more common among Puerto Ricans (7%) than among individuals of Mexican descent (2%). The overall occurrence of FMO2*1 among Hispanics of all origins is estimated to be between 2 and 7%.     

Identification of novel variants of the flavin-containing monooxygenase gene family in African Americans.

Sequence polymorphisms in enzymes involved in drug metabolism have been widely implicated in the differences observed in the sensitivity to various xenobiotics. The flavin-containing monooxygenase (FMO) gene family in humans catalyzes the monooxygenation of numerous N-, P- and S-containing drugs, pesticides, and environmental toxicants. Six genes (FMO1-6) have been identified so far, but the major alleles of FMO2 and FMO6 encode nonfunctional proteins due to a nonsense mutation and splice-site abnormalities, respectively. Data on structural variants exist for human FMO2 and 3, whereas very little is known about the other FMO genes. FMO1-6 were scanned in 50 individuals of African-American descent using the method, detection of virtually all mutations-single-strand conformational polymorphism. A total of 49 sequence variants were identified in a total 1.35 megabases of scanned sequence, of which 29 were variants affecting protein structure or expression. Some of these are expected to affect the activity of the protein, including a nonsense mutation in FMO1 (R502X) and missense mutations in FMO1 (I303T), FMO4 (E339Q), and FMO5 (P457L) that occur in highly conserved amino acids. Additional deleterious substitutions in FMO2 (del337G) and FMO6 (Q105X) were also identified. Multiple structural variants in the FMO gene family were observed in this African-American sample. Some of the substitutions identified in this study might be useful markers in future association studies assessing sensitivity to environmental toxicants and common disease.     

Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones

Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC–MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with K_ms ranging from 7 to 160 μM and turnover numbers of 30–40 min^− 1. The product formed was identified by LC–MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.     

Effect of genetic variants of the human flavin-containing monooxygenase 3 on N- and S-oxygenation activities.

The decreased capacity of the flavin-containing monooxygenase 3 (FMO3) to oxygenate xenobiotics including trimethylamine is believed to contribute to metabolic disorders. The aim of this study was to functionally characterize FMO3 variants recently found in a Japanese population and compare them with selective functional activity of other FMO3 variants. Recombinant Glu158Lys and Glu158Lys-Glu308Gly FMO3 expressed in Escherichia coli membranes showed slightly decreased N-oxygenation of benzydamine and trimethylamine. Selective functional S-oxygenation of these variants by methyl p-tolyl sulfide or sulindac sulfide was comparable to that of wild-type FMO3. The Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val variants showed significantly decreased oxygenation of typical FMO3 substrates (i.e., approximately one-tenth of the V(max)/K(m) values). Val257Met FMO3 had a lower catalytic efficiency for methyl p-tolyl sulfide and sulindac sulfide S-oxygenation. However, compared with wild-type FMO3, Val257Met FMO3 showed a similar catalytic efficiency for N-oxygenation of benzydamine and trimethylamine. The catalytic efficiency for benzydamine and trimethylamine N-oxygenation by Arg205Cys FMO3 was only moderately decreased, but it possessed decreased sulindac sulfide S-oxygenation activity. Kinetic analysis showed that Arg205Cys FMO3 was inhibited by sulindac in a substrate-dependent manner, presumably because of selective interaction between the variant enzyme and the substrate. The results suggest that the effects of genetic variation of human FMO3 could operate at the functional level for N- and S-oxygenation for typical FMO3 substrates. Genetic polymorphism in the human FMO3 gene might lead to unexpected changes of catalytic efficiency for N- and S-oxygenation of xenobiotics and endogenous materials.     

Developmental regulation of flavin-containing monooxygenase form 1 in the liver and kidney of fetal and neonatal rabbits

Flavin-containing monooxygenases (FMOs) comprise a multi-gene family and catalyze the oxygenation of soft nucleophilic sulfur, nitrogen, phosphorus, and selenium in xenobiotics. Previous studies have demonstrated that FMO is regulated developmentally and by the administration of certain steroid hormones. This study examined the expression of FMO form 1 in the livers and kidneys of fetal and neonatal rabbits, from day 25 of gestation through 3 weeks of age, by assaying FMO1 mRNA and protein levels, as well as catalytic activity. FMO1 mRNA and protein expression and FMO catalytic activity were present in fetal livers at the earliest time point measured (day 25 of gestation), although at levels approximately 10% of that found in adult livers. Hepatic FMO1 mRNA levels increased during and after gestation; levels were not significantly different from those measured in adult male livers. FMO1 protein content and activity rose rapidly after birth to reach 70-80% of adult levels by 3 weeks of age. The expression of FMO1 in fetal and neonatal kidneys was markedly lower than in liver. FMO1 mRNA levels never averaged more than 3.4% of adult male liver levels, but did not differ from adult kidney levels at any of the points measured. Protein levels and enzyme activity rose significantly after birth to approximately 30% of the level in adult kidneys by 3 weeks of age. The early developmental appearance of FMO1 suggests a possible role in the metabolism of xenobiotics through transplacental or lactational exposures.     

Substrate specificities of rabbit lung and porcine liver flavin-containing monooxygenases: differences due to substrate size

Phenothiazine, 2-(trifluoromethyl)phenothiazine, and a series of 10-(N,N-dimethylamino-alkyl)-2-(trifluoromethyl)phenothiazines with alkyl side chains varying in length from C2 to C7 were tested for substrate activity with purified rabbit lung and porcine liver flavin-containing monooxygenases (FMO). While all were substrates for the hepatic FMO, only phenothiazines bearing C6 and C7 alkyl side chains were oxidized at significant rates by the pulmonary FMO. Kinetic constants calculated from reaction velocities for the oxidation of thiourea, phenylthiourea, and naphthylthiourea indicate that a nucleophilic heteroatom on the end of a molecule not much larger than a six-membered ring in cross section is oxidized by both enzymes, but the addition of bulky lipophilic substituents increases the Km of N-substituted thioureas for rabbit lung FMO and 1,3-diphenylthiourea (thiocarbanilide) is excluded entirely. From the dimensions of compounds excluded and from those oxidized, it would appear that the hydroperoxyflavin in rabbit lung FMO lies about 6-8 A below the surface in a channel no more than 8 A in diameter in its longest axis. The channel leading to this oxidant in hepatic FMO appears more open and readily admits compounds bearing a tricyclic ring. Differences in dimensions of the substrate channel appear responsible for some of the differences in substrate specificities between liver and lung FMO.     

Mammalian flavin-containing monooxygenase (FMO) as a source of hydrogen peroxide

Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30–50% of O₂ consumed as H₂O₂ upon addition of NADPH. Addition of substrate had little effect on H₂O₂ production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H₂O₂. FMO2 S195L exhibited higher “leakage”, producing much greater amounts of H₂O₂, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H₂O₂ generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H₂O₂ release. Catalase did not reduce levels of H₂O₂ with either FMO2.1 or FMO3 but inhibited H₂O₂ generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP⁺ binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H₂O₂.     

Genetic polymorphisms of human flavin-containing monooxygenase 3: implications for drug metabolism and clinical perspectives

Flavin-containing monooxygenase 3 (FMO3) is a hepatic microsomal enzyme that oxidizes a host of drugs, xenobiotics and other chemicals. Numerous variants in the gene encoding FMO3 have been identified, some of which result in altered enzymatic activity and, consequently, altered substrate metabolism. Studies also implicate individual and ethnic differences in the frequency of FMO3 polymorphisms. In addition, new variants continue to be identified with potentially important clinical implications. For example, the role of FMO3 variants in the pathophysiology of gastrointestinal diseases is an evolving area of research. Two commonly occurring polymorphisms of FMO3, E158K and E308G, have been associated with a reduction in polyp burden in patients with familial adenomatous polyposis who were treated with sulindac sulfide, an FMO3 substrate. These findings suggest a potential role for prospective genotyping of common FMO3 polymorphisms in the treatment of disease states that involve the use of drugs metabolized by FMO3. This review summarizes the current state of research on the genetic polymorphisms of FMO3, with a focus on their clinical implications in gastrointestinal diseases.     

Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: comparative genetic and functional studies.

To characterize the contribution of the human flavin-containing monooxygenase form 3 (FMO3) in the metabolism and disposition of drugs and xenobiotics, we determined the single nucleotide polymorphisms in the coding region and adjacent splice junctions of FMO3 in 134 African Americans and 120 Caucasians from the United States. In the regions examined, DNA resequencing or high throughput MassEXTEND studies coupled with mass spectrometric genotyping showed that 12 sites of variation were present. Three variants encoding synonymous mutations and four polymorphisms were observed in the noncoding region. Another three variants, Lys158-FMO3, Met257-FMO3 and Gly308-FMO3, previously reported in similar populations, were prominent polymorphisms. Two new polymorphisms, His132-FMO3 and Pro360-FMO3, were identified in this study. Both variants were found only in African Americans. To evaluate the effect of the amino acid substitutions on the function of FMO3, each amino acid substitution was introduced by site-directed mutagenesis into a wild-type FMO3 cDNA. Selective functional activity was studied with methimazole, trimethylamine, and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl) phenothiazine. Both His132-FMO3 and Pro360-FMO3 variants were able to metabolize the substrates examined. Compared with wild-type FMO3, the His132-FMO3 was less catalytically efficient. The His132-FMO3 variant moderately altered the catalytic efficiency of FMO3 (decrease of 30%, 60% and 6% with methimazole, trimethylamine and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine, respectively). The Pro360-FMO3 variant was more catalytically efficient than wild-type FMO3. Pro360-FMO3 oxygenated methimazole, trimethylamine and 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine, respectively, 3-, 5- and 2-fold more efficiently than wild-type FMO3. Based on the functional activity of the variant FMO3 enzymes, it is likely that population differences exist for compounds primarily metabolized by FMO3.     

Size limits of thiocarbamides accepted as substrates by human flavin-containing monooxygenase 1.

Microsomes isolated from Spodoptera frugiperda (Sf)9 cells infected with human flavin-containing monooxygenase (FMO)1 recombinant baculovirus catalyzed the NADPH- and O2-dependent oxidation of methimazole, thiourea, and phenylthiourea. However, there was no detectable activity with 1,3-diphenylthiourea or larger thiocarbamides. Microsomes from control Sf9 cells were devoid of methimazole or thiourea S-oxygenase activity. Trimethylamine up to 1.0 mM had no detectable effect on the oxidation of 10 microM methimazole (Km = 5 microM) but 1.0 mM N,N-dimethylaniline or chlorpromazine inhibited the oxidation of 1.0 mM methimazole 50 and 70%, respectively. Although products were not isolated, the pronounced inhibition of methimazole S-oxygenation suggests that these amines are alternate substrates for human FMO1. Because 1,3-diphenylthiourea is apparently completely excluded from the catalytic site, tricyclic amine drugs are probably approaching the upper size limits of xenobiotics accepted by human FMO1. The substrate specificity of this isoform in humans appears considerably more restricted than that of pig or guinea pig FMO1. Differences in the size of nucleophiles accepted must be considered in attempting to extrapolate the extensive structure-activity studies available for pig FMO1 to this FMO isoform in humans.     

Ethnic differences in human flavin-containing monooxygenase 2 (FMO2) polymorphisms: detection of expressed protein in African-Americans

The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.     

Species, organ and cellular variation in the flavin-containing monooxygenase

The distribution of the flavin-containing monooxygenase (EC1.14.13.8) (FMO) between species, organs and cell types is summarized with particular reference to the organ specific forms present in mammalian lung and liver. The role of the FMO relative to cytochrome P-450 in the oxidation of the sulfur atoms of organosulfur compounds is considered with particular reference to the hepatatoxicant thiobenzamide, the insecticide phorate and the drug, thioridazine. Of special interest is the relative role of these enzymes in complex metabolic pathways of xenobiotics.     

含黄素单氧化酶3(FMO3)结构、功能及其基因多态性的研究进展

含黄素单氧化酶3(flavin-containing monooxygenase 3,FMO3)是一种重要的肝微粒体酶,参与体内大量药物、外源性物质和其他一些化学物质的氧化代谢。FMO3基因存在多态性,其中的一些基因突变可以引起酶活性改变,从而改变底物的代谢。体内外许多研究证明FMO3有明显的个体差异和种族差异,因此,FMO3的遗传变异在药物研制和个体化治疗中将起着重要的作用。该文就FMO3结构、功能及其基因多态性与药物代谢和疾病的关系进行综述。     

Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population.

Flavin-containing monooxygenase (FMO) activity was determined in 82 Korean volunteers by taking molar concentration ratio of theobromine and caffeine present in the 1 h urine (between 4 and 5 h) samples collected after administration of a cup of coffee containing 110 mg of caffeine. Among 82 volunteers, there were 19 women and 63 men (30 smokers and 52 non-smokers). Volunteers were divided into two groups comprising low (0.53-2.99) and high (3.18-11.95) FMO activities separated by an antimode of 3.18. Peripheral bloods were sampled from these volunteers and their genomic DNAs were amplified by polymerase chain reaction with oligonucleotides designed from intronic sequences of human FMO3 gene. Comparing nucleotide sequences of the amplified FMO3 gene originating from randomly selected individuals with low and high FMO activities, nine point mutations were identified in the open reading frame sequences. Among these nine mutations, three FMO3 mutant types (FMO3/Stop148, Lys158 and Gly308) were selected and correlated with FMO activities observed in our Korean population. A rare FMO3/Stop148 mutant allele originating from FMO3/Gly148 occurred by substitution of G442T in exon 4 and yielded a premature TGA stop codon. The stop codon was detected in one individual having the second lowest FMO activity and he had the mutation in heterozygous state. In a pedigree study, he was found to have inherited the mutation from his mother who also had a heterozygous stop codon and equally low FMO activity. In our volunteers, two other common mutations were detected in exons 4 and 7. The one in exon 4 resulted from a G472A change eliminating a HinfI restriction site and produced an amino acid substitution from Glu158 to Lys. The other mutation in exon 7 resulted from an A923G change generating a DraII restriction site and produced a non-conservative replacement of Glu308 to Gly. Based on the secondary structure maps of FMO3 enzyme proteins for these two mutant types, FMO3/Gly308 mutation transformed the helix structure into a sheet shape and indicated that dysfunctional FMO3 may be produced. FMO3/Lys158 mutation did not alter the secondary structure. Approximately 80% of volunteers with homozygous and/or heterozygous mutations on either one or two of these mutations had low FMO activities. Thus, individuals with these FMO3 gene mutations may have defective metabolic activity for many clinically used drugs and dietary plant alkaloids which are oxidized primarily by hepatic FMO3.